EP0324834B1 - Verfahren zum schnellen und empfindlichen nachweis von hiv-1-antikörpern - Google Patents

Verfahren zum schnellen und empfindlichen nachweis von hiv-1-antikörpern Download PDF

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Publication number
EP0324834B1
EP0324834B1 EP88906559A EP88906559A EP0324834B1 EP 0324834 B1 EP0324834 B1 EP 0324834B1 EP 88906559 A EP88906559 A EP 88906559A EP 88906559 A EP88906559 A EP 88906559A EP 0324834 B1 EP0324834 B1 EP 0324834B1
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hiv
antibodies
antigen
test
sample
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French (fr)
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EP0324834A1 (de
EP0324834A4 (en
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Kurt B. Osther
Louis M. Dyll
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Verigen Inc
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Verigen Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV

Definitions

  • the present invention relates to a rapid and sensitive assay method for the detection of antibodies to Human T-Cell Leukemia Virus-III (HIV-I) antigen, and diagnostic test kits for carrying out the assay method.
  • HIV-I Human T-Cell Leukemia Virus-III
  • HIV-I HIV-III/LAV
  • HTLV-III/LAV HIV-III/LAV
  • the virus is currently believed to be transmitted through intimate sexual contact and blood. Thus far, epidemiologic evidence indicates that food, water, insects and casual contact are not disease vectors.
  • the AIDS virus acts by crippling the body's immune system.
  • HIV-I selectively attacks T4 lymphocytes, one of the subpopulations of lymphocytes that constitute the immune system.
  • Infection with HIV-I results in both a reduction in the number and a change in the function of the targeted T4 lymphocytes with the eventual collapse of the immune system.
  • the groups at highest risk of infection with HIV-I include homosexual and bisexual men and abusers of injected drugs.
  • Other predictable high risk groups are women artificially inseminated with sperm from infected donors and sexual partners of those in the AIDS risk groups.
  • Recipients of blood transfusions and blood products are also at risk of contracting AIDS.
  • Organ transplant recipients are also a high risk group. Recognizing organ transplantation as a potential avenue for the spread of AIDS, testing for HIV-I antibodies is now performed on all organ donors. With the exception of some kidney transplants it is often difficult to predict in advance the source of a donated organ. For example, victims of fatal automobile accidents are potential organ donors, and as the time and place of accidents are not foreseeable, pre-transplant testing presents a problem. Screening is thus difficult in this and similar situations and the need for 24 hour testing facilities and, more importantly, on sits testing is apparent. Further, in view of the limited useful life of a donated organ once removed from the host, the need for a rapid assay method is evident.
  • the ELISA technique involves reacting a test simple with an antigen reagent generally obtained from disrupted whole or density-banded HIV-I.
  • an antigen reagent generally obtained from disrupted whole or density-banded HIV-I.
  • the HIV-I is coated onto wells of a microtiter plate. After washing to remove unbound antibodies, goat-antihuman IgG antiserum conjugated with horseradish peroxidase is added to the wells and incubated. After an appropriate incubation period, an enzyme substrate is added to the mixture and a detectable, measurable color product is formed in the presence of antibodies to HIV-I.
  • the HIV-I antigen is electrophoretically resolved on SDS-polyacrylamide gels, each 8x10 cm gel being loaded with 50-100 ⁇ g of protein.
  • the resulting protein bands are electro-transferred to nitrocellulose paper.
  • Detection of antibodies to HIV-I is then carried out by either solid phase strip radioimmunoassay techniques or by ELISA. Each of these methods includes an overnight incubation, and thus, an overall test time of about 20 hours.
  • the established screening procedure is not entirely satisfactory because of the time required to obtain results. This is particularly true in the organ transplant situation, especially for the heart and liver. These two organs have maximum cold ischemic times of 4 and 8 hours, respectively. Generally, ELISA takes about 4 hours and the Western Blot which, as noted, includes an overnight incubation period, requires about 20 hours. As can be appreciated, with conventional techniques test results would not be obtained within the maximum ischemic times for the heart or liver.
  • the so-called "Quick Western Blot” assay is a modification of the standard Western Blot assay and is the subject of WO application No. 87/07647.
  • the test involves a Western Blot assay wherein the concentration of the sample being tested as well as the HIV-I antigen used is increased by at least 50% over the standard Western Blot.
  • the technique then involves using the ELISA test to detect the bound antibodies. Because of the increased concentration of the antigen and test sample the resultant assay can be accomplished in as little as one hour and twenty minutes without the expected nonspecific protein noise. Furthermore the test can be done in areas not equipped to perform the other more time consuming tests.
  • the present invention involves a rapid and sensitive method for the detection of antibodies to HIV-I antigen which uses a modified Quick Western Blot technique. Specifically, the method comprises.
  • milk proteins act to both enhance and accelerate specific protein binding; and to decrease unspecific protein binding. Consequently, test results are obtained in 50-60 minutes.
  • the serum sample is diluted in order that non-specific binding of other proteins present therein will not produce overlapping protein banding interfering with the detection of antibodies to the HIV-I antigen.
  • protein concentrations at least 50% greater than those used in the conventional Western Blot have been successfully employed to detect antibodies to the AIDS virus while decreasing the test time by a factor of ten. It has now been found in accordance with the present invention that, when the HIV-I antigen is incubated in the presence of milk proteins, specific binding of the HIV-I antibody to the antigen is both enhanced and accelerated.
  • the concentration of the test sample may be increased relative to that used in the Western Blot Assay without impairing the selectivity of the assay, while the concentration of the sample may be decreased relative to that employed in the Quick Western Blot without either impairing the sensitivity or increasing the incubation times required for the assay.
  • the assay of the present invention results in better resolution than the Western Blot, enhanced specific binding relative to the Quick Western Blot, and markedly decreased incubation times than required to complete either the conventional Western Blot or Quick Western Blot assays.
  • the assay of this invention produces significantly more distinct protein bands. This is extremely important for the reading of the test strips.
  • the milk proteins are present when the antibodies of HIV-I, possibly present in the sample to be tested, are reacted with the HIV-I antigen. It is believed that one or more of the milk proteins changes the electronic structure of the binding site on the antigen thereby increasing its affinity and specificity for the HIV-I antibody.
  • the method of the invention applies blotting techniques but uses at least 20%, preferably about 20% to 40%, more HIV-I viral lysate than conventional Western Blot.
  • at least 20%, preferably about 20% to 40%, more HIV-I viral lysate than conventional Western Blot from 60 to about 120 ⁇ g of HIV-I antigen protein/10x16cm gel is used in the method of the invention, as compared with the 50-100 ⁇ g of protein/10x16cm gel used in conventional Western Blot.
  • Concentrations of test sample are also increased at least 3 times, preferably about 5 to 7 times, than that used in conventional western Blot.
  • the method of the invention like the conventional Western Blot, employs an electrophoretically resolved antigen subsequently electrotransferred to test sheets or strips.
  • test results are obtained in 50-60 minutes, that is 1/30 the time required to perform the Western Blot assay and 3/4 the time required for the Quick Western Blot assay.
  • the method hereof is, therefore, referred to as the "Quick Western Blot II" technique.
  • the method involves electrophoretically resolving HIV-I antigen lysate and subsequently electrotransferring the resolved antigen onto nitrocellulose paper.
  • This paper is incubated with the sample to be tested in order to detect any HIV-I antibodies present.
  • the incubation step is performed in the presence of the milk proteins.
  • nitrocellulose bound antigen/antibody complex is incubated with enzyme conjugated antiserum and developed with an enzyme substrate (color change indicator).
  • enzyme conjugated antiserum enzyme conjugated antiserum
  • developed with an enzyme substrate color change indicator.
  • the resultant colored bands are then visually compared with positive and negative controls in order to ascertain the presence of HIV-I antibodies.
  • a diagnostic test kit which permits on site testing for antibodies to AIDS virus.
  • the kit comprises a set of control tubes for positive and negative references as well as a dilution tube with buffer for dilution of the test sample. The contents of these tubes are added to reaction tubes containing nitrocellulose test strips containing resolved HIV-I antigen and incubated. The resultant strips are incubated with the supplied enzyme conjugated antiserum used to detect whether any bound antibodies are present. The strips are then ultimately developed for color with a color change indicator.
  • the diagnostic kit of the invention also includes predeveloped positive and negative reference strips and reagent control strips for evaluating the test results by visual comparison with the test strips.
  • the visible protein bands on the test sample strip with the positive reference strip should test positive for HIV-I antibodies (as shown from the prespecified protein bands).
  • the negative reference strip should test negative for the HIV-I antibody. Accordingly, reading the results of the test is facilitated and the need for specially trained personnel is virtually eliminated. On site testing is particularly important in the organ transplant situation because screening for AIDS can now be performed on a 24 hour basis at virtually any location, without specially trained personnel, and test results can be obtained in 50-60 minutes, comfortably within the life span of ischemic organs.
  • HIV-I antigen concentrate is electrophoretically resolved.
  • the HIV-I antigen concentrate may be obtained commercially, for example, as from Litton Bionetics as HIV-I viral lysate, Catalog no. 8464-15.
  • the antigen concentrate is diluted in buffer to a protein concentration at least 20% but less than 40% greater than that utilized in conventional Western Blot.
  • the antigen concentrate is diluted in buffer to a protein concentration of about 60 to 120 ug per 10x16cm gel.
  • the preferred buffer is 0.05M TRIS-HCl/50% glycerol, pH8, 2.5% SDS (sodium dodecyl sulfate) and 5% mercaptoethanol.
  • Other buffers known to those skilled in the art are also suitable, such as 9M urea in 0.01M TRIS-HCl.
  • the protein concentration of the antigen lysate used with the method of the invention is approximately 20 to as much as 40% higher than the 50-100ug/10x16cm gel typically used for conventional Western Blot. See pages 380 to 381 of the guidelines published by Tsang V.C., J. Peralta, R. Simons, In: Methods of Enzymology, Vol. 92, Chapter 29, 1983, Academic Press Inc., which sets forth the 50-100 ⁇ g 8x10cm gel workable range of protein concentrations used in the conventional Western Blot assay.
  • the antigen is generally first denatured by boiling, typically for about 5 minutes. Then, the denatured antigen is subjected to conventional gel electrophoresis of the type reported by Tsang et al, Methods in Enzymology, Vol. 92 (1983).
  • a tracking dye is preferably added to the diluted antigen to produce visible protein banding.
  • the preferred dye is bromophenol blue.
  • the dye is preferably prepared by dissolving 50 mg bromophenol blue in 8 ml of glycerol, plus 1 ml each of 0.5M TRIS-HCl at pH 8.0 and H2O.
  • Other dyes known to those skilled in the art, may also be used.
  • Suitable gels for the electrophoresis are also prepared in accordance with the method of Tsang et al., Methods in Enzymology Vol. 92 (1983).
  • a 10% resolving polyacrylamide gel with a 3% stacking gel (SDS-PAGE) is preferred because it resolves a molecular weight range of 12,000-160,000, thus embracing the proteins within the HIV-I viral lysate.
  • SDS-PAGE 10% resolving polyacrylamide gel with a 3% stacking gel
  • a gradient SDS-PAGE gel can also be used.
  • a gradient ranging from 3.3%-20% polyacrylamide gel resolves the antigen in the desired molecular weight range.
  • a molecular weight marker is electrophoresed together with the HIV-I antigen. These marker materials serve to calibrate the gels and facilitate identification of the protein bands of specific molecular weights. Suitable molecular weight markers are commercially available, such as a Cytochrome C molecular weight system from United States Bio
  • the protein bands of the resolved antigen are electro-transferred preferably to nitrocellulose sheets, (e.g., those commercially available from Schleicher and Schuell, Inc. as Item no. BA 83 which is a roll of nitrocellulose paper having a 0.2 ⁇ m pore size).
  • nitrocellulose sheets e.g., those commercially available from Schleicher and Schuell, Inc. as Item no. BA 83 which is a roll of nitrocellulose paper having a 0.2 ⁇ m pore size.
  • Other types of papers known to those skilled in the art, such as diazo-type paper are also suitable.
  • the electro-transfer of the protein bands is accomplished by means of the technique reported by Tsang et al., Methods in Enzymology Vol. 92, particularly page 378, and W. Van Raamsdonk, et al. J. Immunol. Methods 17:337 (1977).
  • the resolved HIV-I protein is incubated in the presence of milk proteins, preferably defatted proteins.
  • milk proteins preferably defatted proteins.
  • Suitable defatted milk proteins included Carnation® lowfat milk powder, but any defatted milk proteins as may be known are also useful.
  • Such milk proteins are known to constitute about 60 to 90% casein, a phosphoprotein rich in serine; and from 10 to 40% of various other proteins including lactoalbumin, lactoglobulin, membrane globulin and a small amount of alkaline phosphatase, peroxidase catalase and xanthine dehydrogenase.
  • the milk proteins may be introduced into the assay system in one of several ways.
  • the nitrocellulose paper blotted with the resolved HIV-I antigen protein is coated with the milk proteins from a solution of buffer (e.g., PBS-Tween® 20), containing about 5 to 10% milk proteins for about 60 minutes.
  • the nitrocellulose paper is subsequently dried at room temperature for a period of 30-60 minutes. After evaporation, the paper is washed in the buffer solution not containing milk proteins (e.g., PBS-Tween® 20) at room temperature For 1-5 minutes. The treated paper is then stored under humid conditions until use.
  • buffer e.g., PBS-Tween® 20
  • the milk proteins are thoroughly mixed with and dissolved in the buffer solutions containing the test sample and controls.
  • the controls and serum samples are then incubated with the nitrocellulose strips For 15 to 20 minutes.
  • the liquid of each tube is discarded and the strips are washed with a PBS-Tween® buffer at pH 7.3-7.4.
  • the washing cycle consists of four 1 minute washings.
  • the milk proteins are precoated on the nitrocellulose strips and additionally mixed with and dissolved in the buffer solution.
  • nitrocellulose sheets are then cut into strips approximately 2-2.5mm in width. Each strip, after appropriate labelling, is placed in a separate test tube or in aluminum foil for determination of antibodies to HIV-I viral lysate by the enzyme linked immunoassay of the invention.
  • the nitrocellulose strips can also be placed in incubation trays for in house testing. It should be understood, however, that an uncut sheet can be placed in an incubation tray equipped with a pressing cover rather than cut into individual strips. This technique is also well-suited to in-house as opposed to on site testing. As can be appreciated, however, on site testing is facilitated by use of individual tubes. Also, placing the strips in individual tubes minimizes the need for handling during the assay procedure and thus, possible smearing of the fragile protein patterns with fingerprints. Using strips is also more economical than uncut sheets because less reagent is necessary to carry out the test.
  • Test samples, positive and negative references and reagent controls are added to the tubes containing the nitrocellulose strips blotted with resolved antigen.
  • Test samples include, but are not limited to serum, semen and other body fluids.
  • the positive reference is typically a sample known to contain antibodies to the HIV-I viral lysate. Positive references have been obtained from the Centers for Disease Control (CDC), Atlanta, Georgia. Alternatively, a positive reference may be made from any sample which has been standardized with a positive reference obtained from the CDC. Standardization typically means that the same test results were obtained in about 20 runs.
  • the positive reference is diluted 1/200 (1 part positive reference to 200 parts buffer) in PBS (phosphate buffered solution)-Tween®, pH 7.2-7.4. Typically, 15 ⁇ l of the positive reference is mixed with 3 ml PBS-Tween®.
  • the negative control is a sample known to be devoid of antibodies to HIV-I viral lysate, and is prepared by diluting 1/20 with PBS-Tween®, pH 7.2-7.4. Typically 150 ⁇ l of a negative reference is mixed with 3 ml PBS-Tween®. Negative references have also been obtained from the CDC.
  • the assay method of the invention is a qualitative rather than quantitative determination
  • the positive and negative references are used to evaluate the test results by comparison with the results obtained for test samples.
  • the reagent control is included as a quality control feature of the present invention and is used to assure accurate functioning of the test.
  • the reagent control is the buffer used to dilute test samples and controls.
  • PBS-Tween®, pH 7.2-7.4 is used as the reagent control.
  • the reagent control is not necessary during routine readings of the strips.
  • test samples may be used which are more concentrated than those used in conventional Western Blot, to accelerate the binding of antibodies to HIV-I viral lysate to the antigen contained in the strips.
  • test samples are about three to five times more concentrated than samples tested by the Western Blot assay.
  • test samples used in accordance with the present invention need to be about 2.5 times less concentrated than the samples employed in the Quick Western Blot assay. The present method thus provides increased sensitivity, without markedly increased non-specific protein binding such as may occur employing very large test sample concentration.
  • the strips are then incubated with the positive and negative references, controls and test samples at room temperature, preferably for about 10 to 20 minutes, to permit the binding of any antibodies to HIV-I present in the sample to the antigen in the nitrocellulose strips.
  • a 20 minute incubation period is particularly preferred to insure optimum binding of weak positives.
  • each tube is discared, with the strips remaining in place in the tubes.
  • the strips are then washed, preferably with PBS-Tween® buffer at pH 7.3.
  • the washing cycle includes four 1 minute washings with PBS-Tween®.
  • the strips are then incubated with an enzyne-conjugated anti-human IgG antiserum preferably for about 10 to 20 minutes, at room temperature, to permit binding of the enzyme conjugated antiserum to any antibody which bound to the antigen during the first incubation period.
  • an enzyne-conjugated anti-human IgG antiserum preferably for about 10 to 20 minutes, at room temperature, to permit binding of the enzyme conjugated antiserum to any antibody which bound to the antigen during the first incubation period.
  • an enzyne-conjugated anti-human IgG antiserum preferably for about 10 to 20 minutes, at room temperature, to permit binding of the enzyme conjugated antiserum to any antibody which bound
  • the washing cycle includes four 1 minute washings with PBS-Tween® followed by one 1 minute washing with PBS.
  • the strips are incubated with an enzyme substrate (color change indicator) for about 10 minutes at room temperature, for production of a color.
  • an enzyme substrate color change indicator
  • the appropriate substrate for use with horseradish peroxidase enzyme is 3,3' diaminobenzidinetetrahydrochloride dihydrate (DAB). It should be understood, however, that substrate selection is dictated by the enzyme used.
  • DAB 3,3' diaminobenzidinetetrahydrochloride dihydrate
  • determination of the presence of antibodies to HIV-I can be accomplished in about 50-60 minutes because of the greatly reduced incubation times, totalling about 40 minutes, as compared with the Western Blot assay which requires at least 20 hours and the Quick Western Blot assay which takes 80 minutes.
  • a self-contained diagnostic test kit which permits "on site" screening for antibodies to HIV-I virus.
  • the test kit includes a set of tubes containing positive and negative references and at least 1 buffer tube containing a predetermined volume of buffer to which the test sample is added in a predetermined amount to obtain a sample concentration from 3 to 7 times greater than that utilized in the Western Blot assay.
  • the reference and control tubes are prediluted, and thus, the user need only dilute the test sample.
  • a set of strip tubes is also provided, each tube containing a nitrocellulose strip containing resolved HIV-I antigen protein, electro-transferred from an SDS-PAGE gel loaded with from 20% to 40% higher antigen protein concentration than that used in conventional Western Blot.
  • the strips contain resolved HIV-I viral lysate, electrotransferred from an 10x16cm SDS-PAGE gel loaded with from 61-96 ⁇ g of antigen protein compared to a range of 51-80 ⁇ g for Western Blot, depending upon the relative amount of specific HIV proteins.
  • the milk protein additive is either pre-dissolved in the buffer solutions containing the test sample and the positive and negative references, or coated on the nitrocellulose strips containing the resolved HIV-I antigen protein.
  • the milk proteins may be separately provided in powder form to be added to the buffer by the user; the resulting buffer solution can then be directly mixed with the test and reference samples, or used to coat the test strips.
  • the reference, control and sample tubes are numbered.
  • the strip tubes are assigned numbers corresponding to those on the reference control and sample tubes.
  • the strips are assigned numbers corresponding to the tubes in which they are placed. This type of numbering system avoids inadvertent mix-ups which can destroy the accuracy of the assay. As can be appreciated, if the top of a tube containing a positive sample is placed on a tube containing a negative sample, it is likely to obtain a false positive result.
  • the kit also contains vials of enzyme-conjugated antiserum reagent, substrate or color change indicator, two washing buffers and solution for terminating the color reaction.
  • enzyme-conjugated antiserum reagent Preferably, goat anti-human IgG antiserum-horseradish peroxidase is used as the enzyme conjugated anti-serum reagent.
  • substrate, reaction terminating agent and washing buffers are DAB, distilled H2O, and PBS-Tween® and PBS, respectively.
  • pre-developed positive and negative reference strips and reagent control strips are provided in the kit. These controls are prepared in substantially the same manner as previously described except that after developing, the strips are air dried. The predeveloped strips are used to evaluate the test results by a visual comparison with the test strips after completion of a color reaction. The reagent control as noted, is provided to assure the accurate functioning of the reagents.
  • the predeveloped reference and control strips are a significant feature of the present invention because they facilitate reading the assay results and practically eliminate the need for a skilled technician to evaluate the results.
  • kit is self-contained, no laboratory equipment is needed.
  • advantages of such a kit are apparent, as it facilitates screening for HIV-I antibodies at any time and virtually at any place, including remote geographic areas and those locations lacking a 24 hour testing facility. As aforementioned, this is of utmost importance in certain organ transplantation situations.
  • gp41 a 41,000 molecular weight protein and especially gp120 and gp160, 120,000 and 160,000 molecular weight proteins, respectively, which are proteins believed to be envelope proteins of the virus.
  • gp41, gp120 and/or gp160 bands are of critical significance in the present method.
  • p24 a 24,000 molecular weight protein.
  • the test is considered positive if activity is recorded at the p24 and gp41 bands and/or the gp120 and/or gp160 bands. Accordingly, proper resolution of the HIV-I antigen lysate is vital.
  • the amount of protein subjected to electrophoresis is related to the distinctiveness of the resulting bands. Obviously, it is desirable to obtain distinct banding at the critical points, particularly, gp20, gp160 and gp41 as well as p24 with few or no noise bands.
  • the method of present invention results in better resolution of the important gp160 and gp120 bands than either of the conventional and Quick Western Blot techniques. The presence of those higher molecular weight proteins provide a more accurate indication of the presence of the AIDS retrovirus in the tested sample since these envelope proteins are considered to be the most reliable bands in the evaluation of the test strips.
  • the protein bands are much clearer and better resolved in the present invention as compared to the other Western Blots, resulting in greater ease in interpreting the results.
  • HIV-I antigen concentrate was electrophoretically resolved in the molecular weight range of 12,000-160,000.
  • the HIV lysate antigen was electro-transferred to nitrocellulose paper. The paper was then cut into strips and placed in separate test tubes.
  • the strips were then incubated with an enzyme conjugated anti human IgG antiserum (1:500) for 15 minutes at room temperature. The liquid was again discarded and the strips were washed four times with PBS-Tween® followed by a one minute washing with PBS alone.
  • the strips were incubated for 10 minutes with horseradish peroxidase and its substrate 3,3' diaminobenzidinetetrahydrochloride dihydrate (DAB).
  • DAB 3,3' diaminobenzidinetetrahydrochloride dihydrate
  • HIV-I antigen concentrate corresponding to 61 ⁇ g/10x16 cm gel was electrophoretically resolved in the molecular weight range of 12,000-160,000.
  • the HIV lysate antigen was electro-transferred to nitrocellulose paper.
  • the paper was then coated in a bath equipped with magnetic stirring with a PBS-Tween® 20 buffer solution containing nonfat milk proteins.
  • the PBS-Tween buffer contained 5% Carnation® milk proteins. This was achieved by mixing 5g of the powder with 100ml of the buffer.
  • the nitrocellulose paper was subsequently dried at room temperature for a period of 5-30 minutes. After evaporation, the paper was washed in PBS-Tween®-20 at room temperature for 2 minutes. The strips were then stored under humid conditions until use.
  • Example I The procedure of Example I was then followed beginning with the initial incubation of the nitrocellulose strips.
  • the presence of antibodies to HIV-I can be detected in 50 minutes.
  • the rapidity with which the test can be performed is enormously important to organ transplant recipients, since donated organs have a limited usable life span outside the body, and in trauma cases where immediate surgery is needed.
  • the Quick Western Blot II through the use of milk proteins, results in both enhanced and accelerated specific protein binding. Furthermore, less unspecific binding of proteins occur.
  • the certainty with which the specific bands can be detected facilitates the diagnosis of AIDS and probably also the provides useful information as to the stage of the HIV infection antibodies to different molecular weight antigen proteins may be indicative of various stages in the progression of the disease. Table II provides information about correlating the presence of specific protein bands with various stages in the progression of the disease.
  • hospitals can obtain fast and accurate results on patients suspected of HIV-I infection, and thus expedite treatment. Moreover, if AIDS is diagnosed, hospital personnel can more readily adopt necessary precautions and minimize accidental viral contamination.

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Claims (16)

  1. Verfahren zum Nachweis von Antikörpern gegen das HIV-I-Retrovirus, umfassend:
    (a) Inkontaktbringen von Nitrozellulosepapier, enthaltend darauf aufgebrachtes aufgetrenntes HIV-I-Antigenprotein, erhalten von einem gelelektrophoretisch aufgetrennten Viruslysat, mit einer Testanalysenprobe, die mit einem Puffer verdünnt ist, und Inkubieren des Nitrozellulosepapiers und der Testanalysenprobe, um die Bindung von Antikörpern, die in der Analysenprobe vorhanden sind, an das Protein auf dem Nitrozellulosepapier zu ermöglichen, worin
    (1) das Viruslysat per Elektrotransfer auf das Nitrozellulosepapier übertragen wird in einer Konzentration von mindestens 20 %, aber weniger als 40 %, größer als die 50-100 µg von HIV-I-Antigenprotein pro 10 x 16 cm Elektrophoresegel, das in einem herkömmlichen Western-Blot-Assay verwendet wird;
    (2) das aufgetrennte HIV-I-Antigenprotein wird in Schritt (a) (1) in der Anwesenheit von fettfreiem Milchprotein inkubiert; und
    (3) die Testanalysenprobe wird in einem Puffer verdünnt auf eine Konzentration, die ungefähr 3-5 mal größer ist als die Konzentration, die in dem herkömmlichen Western-Blot-Assay verwendet wird; oder auf eine Konzentration, die mindestens 3 mal aber weniger als 7 mal größer ist als die 1:100 Verdünnung der Testanalysenprobe, die in dem Western-Blot-Assay im Falle einer Serumanalysenprobe verwendet wird;
    (b) Inkontaktbringen des inkubierten Nitrozellulosepapiers aus Schritt (a) mit einem enzymkonjugierten Antiserum, das mit den Antikörpern reaktionsfähig ist, und Inkubieren, um die Bindung des Antiserums an die Antikörper zu ermöglichen;
    (c) Inkontaktbringen des inkubierten Nitrozellulosepapiers aus Schritt (b) mit einem Enzymsubstrat, das spezifisch ist für das Enzym aus Schritt (b), und Inkubieren, um dadurch eine Farbe zu erzeugen;
    (d) Abstoppen der Farberzeugungsreaktion von Schritt (c); und
    (e) Ermitteln der Menge an hergestellter Farbe als einen Hinweis auf die Anwesenheit von Antikörpern gegen das HIV-I-Viruslysat;
    um den Assay innerhalb von 60 Minuten zu vervollständigen.
  2. Verfahren nach Anspruch 1, worin das fettfreie Milchprotein 60 bis 90 Gew.-% Casein und 10 bis 40 Gew.-% eines Materials umfaßt, das ausgewählt wird aus der Gruppe bestehend aus Lactoglobulin, Membranglobulin, alkalischer Phosphatase, Peroxidasekatalase, Xyanthindehydrogenase und Mischungen davon.
  3. Verfahren nach Anspruch 1, worin das Nitrozellulosepapier, das das aufgebrachte aufgetrennte HIV-I-Antigen enthält, beschichtet wird mit fettfreiem Milchprotein vor der Inkubation mit der Testanalysenprobe in Schritt (a).
  4. Verfahren nach Anspruch 1, worin das fettfreie Milchprotein mit dem Puffer, der zum Verdünnen der Testanalysenprobe verwendet wird, gemischt wird.
  5. Verfahren nach Anspruch 1, worin das Antigenlysat eine Proteinkonzentration zwischen 60 bis 100 µg pro 10 x 16 cm Gel besitzt.
  6. Verfahren nach Anspruch 1, worin die Testanalysenprobe ein Serum ist.
  7. Verfahren nach Anspruch 1, bei dem Schritt (a) wiederholt wird mit mindestens einer Positivkontrolle (einer Analysenprobe enthaltend HIV-I-Antikörper) und mindestens einer Negativkontrolle (einer Analysenprobe, die frei ist von HIV-I-Antikörpern) anstelle der Testanalysenprobe, das dadurch gebildete Reaktionsprodukt wird Schritten (b) bis (e) unterzogen, und die erzeugten Farben werden als Standards mit der Farbe verglichen, die von der Testanalysenprobe erzeugt wird, um die Anwesenheit von Antikörpern gegen das HIV-I-Retrovirus in der Testanalysenprobe zu ermitteln.
  8. Verfahren nach Anspruch 1, worin das aufgetrennte HIV-I-Antigenprotein aus Schritt (a) (1) behandelt wird mit fettfreiem Milchprotein durch
    (1) Beschichten des Nitrozellulosepapiers, hergestellt durch die Übertragung in Schritt (a) (1), mit 3 bis 10 % fettfreiem Milchprotein, oder
    (2) Einbringen von 3 bis 10 % fettfreiem Milchprotein in den in Schritt (a) (2) verwendeten Puffer.
  9. Verfahren nach Anspruch 8, in dem Schritt (a) wiederholt wird mit mindestens einer Positivkontrolle (einer Analysenprobe, enthaltend HIV-I-Antikörper) und einer Negativkontrolle (einer Analysenprobe, die frei ist von HIV-I-Antikörpern) anstelle der Testanalysenprobe, das dadurch gebildete Reaktionsprodukt wird Schritten (b) bis (e) unterzogen, und die erzeugten Farben werden als Standards verglichen mit der von der Testanalysenprobe erzeugten Farbe, um die Anwesenheit von Antikörpern gegen das HIV-I-Retrovirus in der Testanalysenprobe zu ermitteln.
  10. Verfahren nach Anspruch 8, worin die Testanalysenprobe gemischt wird mit einem Puffer ausgewählt aus der Gruppe bestehend aus PBS-Tween, TRIS-Puffern, TBS-Puffern, Harnstoffpuffern und Polyethylenglycol in Kochsalz oder destilliertem Wasser.
  11. Verfahren nach Anspruch 1, worin jeder Inkubationsschritt nicht mehr als 20 Minuten durchgeführt wird.
  12. Verfahren nach Anspruch 11, in dem Schritt (a) wiederholt wird mit mindestens einer Positivkontrolle (einer Analysenprobe, enthaltend HIV-I-Antikörper) und einer Negativkontrolle (einer Analysenprobe, die frei ist von HIV-I-Antikörpern) anstelle der Testanalysenprobe, das dadurch gebildete Reaktionsprodukt wird Schritten (b) bis (e) unterzogen, und die erzeugte Farbe wird als Standards verglichen mit der von der Testanalysenprobe erzeugten Farbe, um die Anwesenheit von Antikörpern gegen das HIV-I-Retrovirus in der Testanalysenprobe zu ermitteln.
  13. Ein diagnostischer Testkit für den Nachweis von AIDS-spezifischen Antikörpern, umfassend:
    (a) einen Satz an Kontrollgefäßen, umfassend positive und negative Referenzen;
    (b) mindestens ein Kontrollreagenzgefäß;
    (c) mindestens ein Verdünnungsgefäß, enthaltend ein vorab bestimmtes Volumen an Puffer zum Verdünnen der Testanalysenprobe, um eine Konzentration der Testanalysenproben von ungefähr 3 bis 5 mal größer als die Konzentration, die in dem Western-Blot-Assay verwendet wird, zu erhalten; oder auf eine Konzentration von 3 bis 7 mal größer als die 1:100 Verdünnung der Testanalysenprobe, die in dem Western-Blot-Assay in dem Fall der Serumanalysenprobe verwendet wird;
    (d) einen Satz an Gefäßen, enthaltend Nitrozelluloseteststreifen, enthaltend aufgetrenntes HIV-I-Antigen, wobei das Antigen von gelelektrophoretisch aufgetrenntem Viruslysat erhalten wurde, worin das aufgetrennte HIV-I-Antigen in einer Konzentration von mindestens 20 % bis 40 % größer ist als die 50 bis 100 µg von Antigenprotein pro 10 x 16 cm Elektrophoresegel, das in dem herkömmlichen Western-Blot-Assay verwendet wird;
    (e) ein fettfreies Milchproteinreagenz zum Binden mit dem aufgetrennten HIV-I-Antigen, wobei das Reagenz gelöst ist in dem Puffer in Verdünnungsgefäß (c) oder beschichtet ist auf die Nitrozelluloseteststreifen in Gefäßen (d);
    (f) ein enzymkonjugiertes Antiserum zum Reagieren mit dem Antikörper-Antigenkomplex;
    (g) ein Farbwechselindikator zum Sicherstellen, ob die HIV-I-Antikörper vorhanden sind; und
    (h) vorentwickelte positive und negative Referenzstreifen und Reagenzkontrollstreifen zum Abschätzen der Ergebnisse des Tests durch visuelles Vergleichen der vorentwickelten Streifen mit den Teststreifen nach dem Beenden einer Farbänderungsreaktion.
  14. Diagnostischer Testkit nach Anspruch 13, worin das elektrophoretisch aufgetrennte Antigen eine Konzentration von 60 bis 100 µg HIV-Protein/10 x 16 cm Gel besitzt.
  15. Diagnostischer Testkit nach Anspruch 13, worin das Verdünnungsgefäß 3 bis 10 % fettfreie Milchproteine enthält.
  16. Diagnostischer Testkit nach Anspruch 15, worin die fettfreien Milchproteine 60 bis 90 Gew.-% Casein und 10 bis 40 Gew.-% eines Materials umfaßt, das ausgewählt wird aus der Gruppe, bestehend aus Lactoglobulin, Membranglobulin, alkalische Phosphatase, Peroxidasekatalase, Xanthindehydrogenase und Mischungen davon.
EP88906559A 1987-07-13 1988-06-28 Verfahren zum schnellen und empfindlichen nachweis von hiv-1-antikörpern Expired - Lifetime EP0324834B1 (de)

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DK362287A DK362287D0 (da) 1987-07-13 1987-07-13 Method for rapid and sensitive detection of hiv-1 antibodies
DK3622/87 1987-07-13
US99311 1987-09-21
US07/099,311 US4885235A (en) 1987-07-06 1987-09-21 Method for rapid and sensitive detection of HIV-1 antibodies
PCT/US1988/002178 WO1989000207A1 (en) 1987-07-13 1988-06-28 Method for rapid and sensitive detection of hiv-i antibodies

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AU637097B2 (en) * 1989-05-09 1993-05-20 Abbott Laboratories Process for preparing an improved western blot immunoassay

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US4452901A (en) * 1980-03-20 1984-06-05 Ciba-Geigy Corporation Electrophoretically transferring electropherograms to nitrocellulose sheets for immuno-assays
US4629783A (en) * 1985-04-29 1986-12-16 Genetic Systems Corporation Synthetic antigen for the detection of AIDS-related disease
DE3722272A1 (de) * 1986-07-08 1988-01-21 Bio Rad Laboratories Differenzierung des krankheitszustandes bei aids-antikoerpertests
US4865966A (en) * 1987-04-17 1989-09-12 New York University Method for detecting antibodies to human immunodeficiency virus

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JOURNAL OF INFECTIOUS DISEASES, vol. 155, no. 1, January 1987; K. MARTIN et al., pp. 54-63# *
METHODS OF ENZYMOLOGY, vol. 92, chapter 29, Academic Press, 1983; TSANG et al., pp. 377-391# *

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WO1989000207A1 (en) 1989-01-12
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EP0324834A1 (de) 1989-07-26
EP0324834A4 (en) 1992-05-06

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